Cytotoxicity of Sanguinarine Chloride to Cultured Human Cells from Oral Tissue

Abstract: The in vitro cytotoxicity of sanguinarine chloride, a dental product used in the treatment of gingivitis and plaque, was compared using cell lines and primary cells from oral human tissues. For the established cell lines, sanguinarine chloride exhibited similar potencies to S-G gingival epithelial cells and to KB carcinoma cells, whereas HGF-1 gingival fibroblasts were more tolerant. However, a gingival primary cell culture was more sensitive to sanguinarine chloride than were the established cell lines. Detailed studies were performed with the S-G cells. The 24-hr midpoint (NR₅₀) cytotoxicity value towards the S-G cells was 7.6 uM, based on the neutral red cytotoxicity assay; vacuolization and multinucleation were noted. When exposed to sanguinarine chloride for 3 days, a lag in growth kinetics was first observed at 1.7 μM. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 3 hr exposure to sanguinarine chloride at 0.1275 mM and greater. The cytotoxicity of sanguinarine chloride to the S-G cells was lessened in the presence of an S9 hepatic microsomal fraction from Aroclor-induced rats or by including fetal bovine serum (15%) in the exposure medium. Progressively increasing the pH from 6.0 to 7.8 enhanced the potency of sanguinarine chloride, presumably due to the enhanced uptake of the lipophilic alkanolamine form, as compared to that of the cationic iminium form.     

Characterization of the acetaminophen-induced degradation of cytochrome P450-3A4 and the proteolytic pathway

This study compared the in vitro responses of malignant and normal cells from the human oral cavity to tea extracts and to its main polyphenolic component, (-)-epigallocatechin gallate (EGCG). The antiproliferative effects of tea polyphenolic extracts and EGCG were more pronounced towards immortalized, tumourigenic (CAL27, HSC-2, and HSG(1)) and non-tumourigenic (S-G) cells than towards normal (GN56 and HGF-1) fibroblasts and green tea was more toxic than black tea. As the addition of tea extract or EGCG to cell culture medium led to the formation of hydrogen peroxide (H(2)O(2)), the research then focused on EGCG as an inducer of oxidative stress, using CAL27, the cancerous cells most sensitive to EGCG, HSG(1), the cancerous cells least sensitive to EGCG, and GN56 cells. The toxicity of EGCG was decreased in the presence of catalase, an enzyme that degrades H(2)O(2), or of deferoxamine, a chelator of Fe(3+). Conversely, pretreatment of the cells with the glutathione depleters, 1-chloro-2,4-dinitrobenzene and 1,3-bis(2-chloroethyl)-N-nitrosourea, potentiated the toxicity of EGCG. A 4-hr exposure to EGCG lessened the intracellular level of reduced glutathione in the CAL27 and HSG(1) cells, but not in the GN56 fibroblasts. Whereas EGCG itself did not induce lipid peroxidation, Fe(2+)-induced lipid peroxidation was potentiated by EGCG. A 72-hr exposure to cytotoxic concentrations of EGCG induced significant cytoplasmic vacuolization in all cell types. The results presented herein are consistent with EGCG acting as a prooxidant, with the cancerous cells more sensitive to oxidative stress than the normal cells.     

In vitro cytotoxicity of (-)-catechin gallate, a minor polyphenol in green tea

The cytotoxicity of (-)-catechin gallate (CG), a minor polyphenolic constituent in green tea, towards cells derived from tissues of the human oral cavity was studied. The sequence of sensitivity to CG was: immortalized epithelioid gingival S-G cells>tongue squamous carcinoma CAL27 cells>salivary gland squamous carcinoma HSG cells>normal gingival HGF-1 fibroblasts. Further studies focused on S-G cells, the cells most sensitive to CG. The response of the S-G cells to CG was dependent on the length of exposure, with midpoint cytotoxicity values of 127, 67 and 58muM CG for 1-, 2- and 3-day exposures, respectively. The sequence of sensitivity of the S-G cells to various green tea catechins was characterized as follows: CG, epicatechin gallate (ECG)>epigallocatechin gallate (EGCG)>epigallocatechin (EGC)>epicatechin (EC), catechin (C). The cytotoxicity of CG, apparently, was not due to oxidative stress as it was a poor generator of H(2)O(2) in tissue culture medium, had no effect on the intracellular glutathione level, its cytotoxicity was unaffected by catalase, and it did not induce lipid peroxidation. However, CG did enhance Fe(2+)-induced, lipid peroxidation. CG-induced apoptosis was detected by nuclear staining, both with acridine orange and by the more specific TUNEL procedure. The lack of caspase-3 activity in cells exposed to CG and the detection of a DNA smear, rather than of discrete internucleosomal DNA fragmentation, upon agarose gel electrophoresis, suggest, possibly, that the mode of cell death was by a caspase-independent apoptotic pathway. The overall cytotoxicity of CG was similar to its epimer, ECG and both exhibited antiproliferative effects equivalent to, or stronger than, EGCG, the most abundant catechin in green tea.     

In vitro cytotoxicity of a theaflavin mixture from black tea to malignant, immortalized, and normal cells from the human oral cavity.

The growth inhibitory effects of a theaflavin mixture from black tea were more pronounced to malignant (CAL27; HSC-2; HSG1) and immortalized (S-G; GT1) cells than to normal (HGF-2) cells from the human oral cavity. Studies with malignant carcinoma CAL27 cells and immortalized GT1 fibroblasts showed that cytotoxicity of the theaflavin mixture was enhanced as the exposure time was increased, with the tumor CAL27 cells more sensitive than the GT1 cells. Hydrogen peroxide (H(2)O(2)) was detected in cell culture medium amended with the theaflavin mixture. The level of H(2)O(2) in cell culture medium amended with the theaflavin mixture was lessened in the presence of catalase and CoCl(2); the level of authentic H(2)O(2) was also lessened in the presence of CoCl(2), suggesting that Co(2+) led to the rapid catalytic decomposition of H(2)O(2). The cytotoxicity of the theaflavin mixture was due, in part, to the generation in the cell culture medium of H(2)O(2), which lessened the intracellular levels of glutathione in the CAL27 cells and, to a lesser extent, in the GT1 cells. For both cell types, coexposures of the theaflavin mixture with catalase or CoCl(2) afforded protection.     

In vitro cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine, towards cells from human oral tissue.

The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D,L-penicillamine, the SNAP metabolites, N-acetyl-D,L-penicillamine disulfide and nitrite, and preincubated (denitrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>spermine NONOate> SNAP>DPTA NONOate>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

In vitro cytotoxicity of glyco-S-nitrosothiols. a novel class of nitric oxide donors.

The cytotoxicities of the nitric oxide (NO) donors, S-nitroso-N-acetylpencillamine (SNAP) and three glyco-SNAPs, glucose-1-SNAP, glucose-2-SNAP, and fructose-1-SNAP, towards the human gingival epithelioid S-G cell line and three human carcinoma cell lines derived from tissues of the oral cavity were compared using the neutral red (NR) assay. In general, the glucose-SNAPs were more cytotoxic than SNAP, which, in turn, was more cytotoxic than fructose-1-SNAP. Further studies focused on the response of S-G cells to glucose-2-SNAP. The cytotoxicity of glucose-2-SNAP was attributed to NO, as glucose-2-SNAP (t1/2=20 h at 28 degrees C) aged for 4 days was nontoxic, toxicity was eliminated in the presence of hydroxocobalamin, a specific NO scavenger, and toxicity was not noted with glucose-2-AP (the parent compound used to construct glucose-2-SNAP). Exposure of cells to glucose-2-SNAP resulted in a lessening of the intracellular level of glutathione and cells pretreated with the glutathione-depleter, 1,3-bis-(chloroethyl)-1-nitrosourea, were more sensitive to a subsequent challenge with glucose-2-SNAP. Cytotoxicity of glucose-2-SNAP was lessened upon coexposure with the antioxidants, myricetin, N-acetyl-L-cysteine, and L-ascorbic acid. S-G cells exposed to glucose-2-SNAP exhibited bi- and multinucleation. Death of S-G cells exposed to glucose-2-SNAP apparently occurred by apoptosis, as demonstrated with fluorescence microscopy by the appearance of brightly stained, hypercondensed chromatin in spherical cells and of membrane blebbing and by the DNA-ladder of oligonucleosome-length fragments noted with gel electrophoresis. In comparison with other classes of NO donors the sequence of toxicity towards S-G cells was S-nitrosoglutathione>glucose-SNAPs>SNAP, sodium nitroprusside>spermine NONOate>DPTA NONOate>DETA NONOate>fructose-1-SNAP>SIN-1.     

A comparative study on subacute toxicity of arsenic trioxide and dimethylarsinic acid on antioxidant status in Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC) cell lines

Arsenic (As) is a global contaminant of terrestrial and aquatic environments posing concern for environmental and human health. The effects of subacute concentrations of arsenic trioxide (As^III) and dimethylarsinic acid (DMA^V) were examined using Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC). Whole monolayer with suffering cells (confluence 100%, pyknosis and refractive cells; value scale = 2) led to identification of subacute As concentrations for the three cell lines. The selected As^III concentrations were 1.33 µM for CRFK and 33.37 µM for PLC/PRF/5 and EPC, at 48 hr time point. The selected DMA^V concentrations were 0.67 mM for PLC/PRF/5, 1.33 mM for CRFK, and 2.67 mM for EPC for 48 hr. Unlike the As^III test, the three cell lines did not exhibit marked susceptibility to DMA^V-mediated toxicity. Several oxidative stress biomarker levels, directly or indirectly associated with reactive oxygen species (ROS) elimination including superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, and total glutathione, were determined in the three cell lines at 24 and 48 hr. Antioxidant responses in metal-treated cells were significantly altered compared to controls, suggesting a perturbation of redox state. The weakening of antioxidant pathway in either healthy or tumoral cells was greater using As^III than DMA^V. Differences in level of several oxidative stress biomarkers suggest that the oxidative stress mechanism induced by As^III is distinctly different from DMA^V. Multifaceted mechanisms of action underlying ROS generation in tumor and nontumor cells versus As^III and DMA^V exposure are thus involved. Since As-mediated toxicity is quite complex, more data regarding both oxidant-enhancement and oxidant-lowering strategies may be useful to improve knowledge regarding the influence of As on human and animal cells.     

In vitro Cytotoxicity of the Nitric Oxide Donor,S-Nitroso-N-acetyl-peraciUamine,towards Cells from Human Oral Tissue

Abstract: The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D, L-penicilIamine, the SNAP metabolites, N-acetyl-D, L-penicillamine disulfide and nitrite, and preincubated (denilrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>>spermine NONOate> SNAP>DPTA NONOate>>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1, 3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP. Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

Free fatty acids enhance the oxidative damage induced by ethanol metabolism in an in vitro model

In recent years, there has been a growing interest to explore the responsiveness to injury in steatotic hepatocyte. VL-17A cells, which express ADH and Cyp2E1 overloaded with free fatty acids (1 mM of oleic and palmitic acid 2:1) showed an increased oxidative damaged after 24 h free fatty acids treatment when exposed to ethanol (100 mM) for 48 h as a second injury. An increment in reactive oxygen species, determined by DCFH-DA, protein oxidation, and apoptosis were observed although an increase in main antioxidant proteins such as superoxide dismutase 1 and glutathione peroxidase were observed, but failed in gamma-glutamylcysteine synthetase, suggesting a decreased capacity of synthesis of glutathione compared with cells treated only with free fatty acids or ethanol. The increased oxidative stress and toxicity in lipid overloaded VL-17A cells subjected to ethanol exposure were accompanied by increases in Cyp2E1 protein expression. Our data show that lipid loaded in an in vitro model, VL-17A cells, is more susceptible to cell damage and oxidative stress when treated with ethanol.     

Effect of lead acetate on cytosolic thioredoxin reductase activity and oxidative stress parameters in rat kidneys

Oxidative stress has been suggested to be an important molecular mechanism of toxic effects of lead in the kidney. Thioredoxin reductase-1 is a selenoprotein involved in many cellular redox processes. This study evaluated the effect of acute and chronic exposure intraperitoneally to lead acetate on thioredoxin reductase-1 activity and on other oxidative stress parameters in the rat kidney, as well as on indicators of renal function commonly used to assess lead poisoning. Acute exposure to 25 mg/kg lead acetate increased superoxide dismutase and thioredoxin reductase-1 activity (after 6, 24 and 48 hr), while exposure to 50 mg/kg lead acetate increased catalase activity (after 48 hr) and inhibited delta-aminolevulinate dehydratase activity (after 6, 24 and 48 hr) in the kidney (P < 0.05). Chronic exposure (30 days) to 5 mg/kg lead acetate inhibited delta-aminolevulinate dehydratase and increased glutathione S-transferase, non-protein thiol groups, catalase, thioredoxin reductase-1 and uric acid plasma levels, while exposure to 25 mg/kg lead acetate reduced body weight and delta-aminolevulinate dehydratase, but increased glutathione S-transferase, non-protein thiol groups and uric acid plasma levels (P < 0.05). No changes were observed in thiobarbituric acid reactive substances, glutathione peroxidase, creatinine or inorganic phosphate levels after either acute or chronic exposure. Our results suggest that thioredoxin reductase-1 may be an early indicator of acute exposure to low lead doses.     

Paraquat-induced oxidative stress and dysfunction of the glutathione redox cycle in pulmonary microvascular endothelial cells

Oxidative stress and changes in the antioxidant defense system that include the glutathione redox cycle in cultured pulmonary microvascular endothelial cells after exposure to paraquat at 0.1 and 0.5 mM were examined as a function of time. Cell viability was substantially lost 72 h after exposure to 0.5 mM paraquat, but not 0.1 mM paraquat. Viability loss was accompanied by increased glutathione-protein mixed disulfide formation, as well as a loss in glyceraldehyde-3-phosphate dehydrogenase activity, indicating a low defense potential. At 4 h after exposure to paraquat at both doses, however, a marked loss in NADPH was found, together with a decrease in aconitase activity. With 0.5 mM paraquat, increased NADP(+) accompanied by NADPH loss diminished constantly after 48 h without recovery of lost NADPH, suggesting destruction of pyridine nucleotides under oxidative stress. NAD(+) decreased 72 h after exposure to 0.5 mM paraquat, but NADH was not influenced. 3-Aminobenzamide did not protect the loss in NADP(+) or NAD(+) and cell viability. Although oxidized glutathione did not increase by exposure to paraquat at both doses through a 96-h exposure period, reduced glutathione increased at 48 to 72 h, with an increase in glutathione disulfide reductase activities. In contrast, a marked loss in glutathione peroxidase activity was produced 48 h after exposure to 0.5 mM paraquat, preceding cell injury. Mercaptosuccinate, an inhibitor of glutathione peroxidase, distinctly hastened viability loss by paraquat. These results indicate that the reduced ability of the glutathione redox cycle, leading to high oxidative stress, is closely associated with paraquat-induced cytotoxicity.     

Protective effect of protocatechuic acid from Alpinia oxyphylla on hydrogen peroxide-induced oxidative PC12 cell death

The neuroprotective effects of protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, on hydrogen peroxide (H(2)O(2))-induced apoptosis and oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to 0.4 mM H(2)O(2) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCA increased PC12 cellular viability and markedly attenuated H(2)O(2)-induced apoptotic cell death in a dose-dependent manner. By flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against H(2)O(2)-induced apoptosis. In these cells, the levels of glutathione (GSH) and activity of catalase were augmented, while glutathione peroxidase activity remained unchanged. In addition, PCA also protected against cell damage induced by H(2)O(2) and Fe(2+), which generated hydroxyl radicals (OH) by the Fenton reaction. These results suggest that PCA may be a candidate chemical for the treatment of oxidative stress-induced neurodegenerative disease.     

Relationships between ascorbic acid and alpha-tocopherol during diquat-induced redox cycling in isolated rat hepatocytes

The effects of diquat-induced redox cycling on the levels of cellular ascorbic acid and alpha-tocopherol were investigated in isolated rat hepatocytes. In untreated hepatocytes, the metabolism of 1 or 2 mM diquat resulted in the depletion of cellular ascorbic acid and glutathione, but not of alpha-tocopherol, in association with the induction of cell death during the experimental period. In 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) pretreated cells, 1 mM diquat induced cell death accompanied by glutathione was rapid (to 9% of controls by 15 min) and cell ascorbate was completely consumed by 2 hr of incubation. In contrast, cellular alpha-tocopherol levels were stable for the first 30 min, but were depleted in association with the onset of lipid peroxidation. Supplementation of 0.1 or 1.0 mM ascorbic acid in the incubation medium delayed the onset of diquat-induced alpha-tocopherol loss, lipid peroxidation and cytotoxicity. When the concentration of exogenous cellular ascorbic acid was consumed to below that of endogenous ascorbic acid, alpha-tocopherol loss and lipid peroxidation were initiated. The results indicate that untreated hepatocytes have an effective multicomponent antioxidant system against diquat-induced oxidative stress. However, when glutathione is depleted from hepatocytes by treatment with BCNU and diquat, ascorbic acid plays a vital role in maintaining cellular alpha-tocopherol levels and survival of the cell.     

Toxicity of aromatic thiols in the human red blood cell

Thiophenol and 4-aminothiophenol were used to study levels of toxicity in human red blood cells. Thiophenols caused conversion of oxyhemoglobin to methemoglobin. Reduction of corresponding disulfides by intracellular glutathione caused cyclic reduction/oxidation reactions, resulting in increased oxidative flux. Three levels of oxidative stress were observed in these experiments: the lowest level resulted from incubation with 0.25 mM thiophenol; the intermediate level with 0.50 mM thiophenol or 0.25 mM 4-aminothiophenol; the highest levels with 0.50 mM 4-aminothiophenol. Methemoglobin formation increased with increasing level of oxidative stress. Glycolysis and the hexose monophosphate shunt were inhibited at the intermediate and highest levels of stress, respectively. Above the highest level of stress non-intact hemoglobin was formed and cell lysis occurred. These metabolic responses were reflected in cellular levels of NADH, NADPH and reduced glutathione. At the lowest level of oxidative stress, both glycolysis and hexose monophosphate shunt were increased such that near-normal levels of NADH, NADPH and reduced glutathione were maintained and methemoglobin formation was kept to a minimum. The response of red cells to 0.25 mM thiophenol appears to represent a level of oxidative stress to which the cell is capable of adaptive metabolic response. Glycolysis contributes approximately one-quarter of the total reducing equivalents from glucose metabolism in response to the oxidative challenge by thiophenol. The results suggest that the metabolic response to autoxidation of endogenous thiols is thiol exchange with glutathione and reduction of resulting glutathione disulfide by the hexose monophosphate shunt.     

In vitro response of human gingival epithelioid S-G cells to minocycline.

Minocycline, a broad-spectrum antibiotic used in the treatment of acne and periodontal disease and to control inflammatory diseases such as rheumatoid arthritis, has recently been shown to induce a spectrum of adverse health effects. In the light of these contradictory data, this research was directed to provide basic information on the toxicology of minocycline, using in vitro cell culture models, and to evaluate its efficacy in periodontal therapies, particularly for wound healing. The human gingival epithelioid S-G cell line was used as the bioindicator. The greater toxicity of minocycline over doxycycline and tetracycline, related antimicrobial agents, probably correlated with its higher lipophilicity. The cytotoxicity of minocycline was unaffected by an S9 hepatic microsomal fraction, indicating that it is a direct-acting, rather than a metabolism-mediated, cytotoxicant. In comparative toxicity studies, much variation in the degree of sensitivity to minocycline was noted for different cell types. No correlation in the extent of sensitivity to minocycline and the physiologic state of the bioindicator cell (normal, transformed or malignant) was noted. The toxicity of minocycline to the S-G cells was dependent on its concentration and length of exposure. For a continuous 3-day exposure of the S-G cells to minocycline, the midpoint cytotoxicity (or, NR(50)) value, as quantified in the neutral red (NR) assay, was 204 microg/ml on day 1, 84 microg/ml on day 2, and 59 microg/ml on day 3. For a 1-h exposure of the S-G cells in phosphate buffered saline (PBS), the NR(50) value was 780 microg/ml minocycline. Although a 1-h exposure in PBS to 200 microg/ml minocycline exerted some toxicity, the S-G cells recovered on exposure to growth medium; irreversible, progressive damage occurred at 400 microg/ml minocycline and greater. Minocycline, at 50 microg/ml, enhanced attachment of the S-G cells to a gelatin-coated surface and cell migration towards an immobilized fibronectin gradient, both biologic parameters important in periodontal wound healing. Minocycline generally had little or no effect on production of the pro-inflammatory cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), by non-activated S-G cells, the exception being stimulation of IL-6 at 48 h. IL-1beta, however, greatly stimulated IL-6 and IL-8 production, which was further increased by concurrent exposure to minocycline. This suggested that minocycline may enhance the ability of gingival epithelial cells to participate in the early, inflammatory phase of periodontal wound healing. The limitation of minocycline efficacy to a rather narrow window of concentration, centering about 50 microg/ml, and primarily for short-term exposures may possibly explain, in part, the contradictory clinical data on the health effects of this drug.     

The role of glutathione in the regulation of nucleotide excision repair during oxidative stress

Nucleotide excision repair (NER) mainly repairs bulky DNA adducts and helix distorting lesions, but is additionally considered to be a back-up system for base excision repair to remove oxidative stress induced DNA damage. Therefore, it can be speculated that NER is up-regulated or primed by oxidative stress. Exposure of human pulmonary epithelial cells (A549) to non-toxic doses of 100muM H(2)O(2) indeed showed a 2 to 4.5-fold increase in expression of XPA, XPC, ERCC4, and ERCC5, whereas the expression of ERCC1 was 5-fold decreased. Phenotypical assessment of NER capacity (i.e. recognition and incision of benzo[a]pyrene-DNA adducts) showed a significant decrease to less than 50% after H(2)O(2) exposure, which paralleled the effects of H(2)O(2) on ERCC1 expression. To study the possible involvement of glutathione (GSH) in the regulation of NER, cells were pre-incubated with 0.5mM BSO, resulting in total GSH depletion and increased intracellular oxidative stress. In GSH-depleted cells, the down-regulation of ERCC1 expression by H(2)O(2) was completely abolished and the up-regulation of ERCC4 expression was potentiated from 2.5-fold to >10-fold. Similarly, the H(2)O(2)-induced decrease in NER capacity was absent in GSH-depleted cells. Overall, our data suggest that NER capacity as well as the expression of NER related genes can be modulated by oxidative stress. ERCC1 expression and NER capacity correlated strongly (R(2)=0.85, P<0.01) after oxidant exposure, indicating ERCC1 as a specific target for oxidative stress induced modification of NER.     

N-acetylcysteine does not protect HepG2 cells against acetaminophen-induced apoptosis

Acetaminophen in large doses is well-known as hepatotoxic, and early therapy with N-acetylcysteine is frequently life-saving. However, in later stages of acetaminophen poisoning, treatment with N-acetylcysteine is not always effective. Although some of the pathways of acetaminophen toxicity and the effect of N-acetylcysteine have been elucidated, in depth information on this process is still lacking. Hepatoma-derived HepG2 cultured cells were exposed to acetaminophen (5 and 10 mM), with or without N-acetylcysteine (5 mM), for 24 and 48 hr. For the assessment of oxidative damage, apoptosis and necrosis, we followed redox status, glutathione content, nuclear fragmentation, phosphatidylserine externalization and ultrastructural changes. Variations in Ca2+ level and number of mitochondrial dense granules were also studied. Acetaminophen treatment of HepG2 cells caused oxidative damage and apoptosis. Significant decrease of cellular redox potential and glutathione content were time- and concentration-dependent. The protective effect of N-acetylcysteine was expressed by an increase of intracellular glutathione and of the level of metabolic reduction of the redox indicator Alamar Blue. The apoptogenic effect of acetaminophen was assessed by flow cytometry of annexin V binding, nuclear hypodiploidity, intracellular Ca2+, as well as by ultrastructural examination. Beyond 24 hr of acetaminophen exposure, necrosis was also noticed. We conclude that acetaminophen-induced oxidative damage in HepG2 cultured cells can be prevented by exposure to N-acetylcysteine. However, apoptosis, either early or late, here demonstrated, is not avoided by exposure to N-acetylcysteine. N-Acetylcysteine did not prevent acetaminophen-induced plasma membrane asymmetry, nuclear damage, alterations of Ca2+ homeostasis and ultrastructural changes.     

In vitro cytotoxicity to human cells in culture of some phenolics from olive oil

The neutral red in vitro cytotoxicity assay was used to evaluate the comparative responses of human cells isolated from tissues of the oral cavity to olive oil phenolics. The cell lines used included normal gingival fibroblasts, immortalized, nontumorigenic gingival epithelial cells, and carcinoma cells from the salivary gland. No differences in the relative sensitivities to the phenolics amongst the three cell types were noted. In general, for all cell types, the sequence of increasing cytotoxicity was: oleuropein aglycone>oleuropein glycoside, caffeic acid>o-coumaric acid>cinnamic acid>tyrosol, syringic acid, protocatechuic acid, vanillic acid. Cytotoxicity was noted only at phenolic concentrations far exceeding those attainable after habitual consumption, thus indicating that consumption of phenol-rich olive oil is safe.     

Antioxidants do not prevent acrylonitrile-induced toxicity

Several reports have recently described that acrylonitrile (ACN) toxicity resides in its capacity for inducing oxidative stress. ACN can be conjugated with glutathione (GSH), diminishing its cellular content, or being metabolized to cyanide. In the present report, we determine the effect of ACN on the viability of primary-cultured astrocytes as well as the oxidative damage generated by ACN by measuring GSH levels in primary cultured astrocytes. We also analyzed whether the ACN (2.5mM) toxicity could be avoided by using antioxidants such as taurine (5mM), N-acetylcysteine (20 mM), trolox (100 microM), estradiol (10 microM) and melatonin (100 nM-1mM). In this cell culture model, antioxidants were not able to prevent ACN-induced cell damage, with the exception of NAC, confirming that only GSH seems to play a key role in ACN-derived toxicity. Additionally, we measured different parameters of oxidative stress such as catalase activity, lipid peroxidation and GSH concentration, as indicators of the potential oxidative stress mediated by the toxicity of ACN, after exposure of Wistar rats to a concentration of 200 ppm ACN for 14 days. At the concentration assayed, we did not find any evidence of oxidative damage in the brain of ACN-treated rats.